Retinoblastoma is a malignant tumor of the sensory layer of the retina. The neoplastic tumor is composed of primitive retinal cells, occurring often bilaterally, usually before the third year of life. It exhibits a familial tendency. Retinoblastomas are characterized by small round cells with deeply staining nuclei, and elongated cells forming rosettes. They usually cause death by local invasion, especially along the optic nerves.
The retinoblastoma may be hereditary but also acquired. It is the most common intraocular tumor and represents one of the prototypes of inheritable cancers. The hereditary form is characterized by early age of onset and multiple tumor foci. Acquired form occurs later in life with single unilateral tumor (Proc. Natl. Acad. Sci., 68:820-823 [1971]; Hum. Genet., 52:1-54 [1979]; Science, 223:1028-1033 [1984]).
The molecular mechanism of the formation of this tumor is unknown. Absence or inactivation of the retinoblastoma (RB) gene is believed to be the primary cause of this cancer (Science, 213:1501-1503 [1981]; Science, 223:1028-1033 [1984]; Proc. Natl. Acad. Sci., 68:820-823 [1971]; Nature, 305:779-784 [1980]).
Susceptibility to hereditary retinoblastoma is transmissible to offsprings as an autosomal dominant trait with 90% penetrance, and the tumor is, therefore, a prototypic model for the study of genetic determination in cancer (Am. J. Hum. Genet., 30:406-410 [1978]; Cancer, 35:1022-1026 [1975]).
There are at least two hypotheses related to the oncogenesis of retinoblastoma. The first hypothesis suggests that the tumor is caused by two mutational events (Proc. Natl. Acad. Sci., 68:820-823 [1971]; Cancer, 35:1022-1026 [1975]). The other hypothesis proposes that autosomal dominant hereditary tumors, such as retinoblastoma, represent the inheritance of a defective regulatory or suppressor gene which normally regulates a group of transforming genes, most probably protooncogenes. Such genes, when active, would release the cell from its normal constraints on growth and therefore, if and when somatic mutation inactivates the normal suppressor gene, such as for example the RB gene, tumors can develop (Proc. Natl. Acad. Sci., 70:3324-3328 [1973]).
Based on these hypotheses, hereditary retinoblastoma might arise from a precursor retinoblast cell, carrying one inherited defective allele which suffers an additional somatic mutation, while nonhereditary cases would require two somatic mutations in the same cell. Recent circumstantial evidence supports the existence of such cancer suppressor genes in retinoblastoma (Nature, 305:779-781 [1983]; Proc. Natl. Acad. Sci. 83:7391-7394 [1986]; Science, 235:1394-1399 [1987]) as well as nephroblastoma also known as Wilm's tumor (Nature, 309:172-174 [1984]; Nature, 309:176-178 [1984]; neuroblastoma (Cancer Res., 41:4678-4682 [1981]), osteosarcoma (Proc. Natl. Acad. Sci., 82:6216-6220 [1985]), ductal breast carcinoma (Proc. Natl. Acad. Sci., 84:2372-2376 [1987]), and other tumors (Nature, 322:644-647 [1986]). However, the molecular nature of this regulation is unknown at the present time.
Cytogenetic studies done previously indicate that deletions of human chromosome 13 band q14 were occasionally found in somatic cells from patients with retinoblastoma (Hum. Genet., 52:1-54 [1979]; Ann. Hum. Genet., 27:171-174 [1963]; Am. J. Dis. Child, 132:161-163 [1978]; Science, 208:1042-1044 [1980]; Science, 213:1501-1503 [1981]). Linkage was established to the gene for the polymorphic marker enzyme esterase D, which also maps to 13q14 (Science, 219:971-973 [1983]).
Additional evidence supporting this genetic assignment came from the pedigree of a family showing balanced chromosomal translocations in unaffected carrier parents and in some unaffected siblings but with an unbalanced chromosome 13 deletion in affected individuals (Science, 213:1501-1503 [1981]). This observations also indicated that the retinoblastoma susceptibility locus is able to function in the "trans" configuration.
The RB locus was further implicated in non-hereditary retinoblastoma by observing frequent abnormalities of chromosome 13 in tumor karyotypes and reduced esterase D activity in tumors (Cancer Genet. Cytogenet., 10:311-333 [1983]; Cancer Genet. Cytogenet., 6:213-221 [1982]). It has been proposed that inactivation of both alleles of the RB gene located in region 13q14 resulted in retinoblastoma. Such proposal was based in part on a case of hereditary retinoblastoma in which both RB alleles were inferred to be absent (Science, 219:973-975 [1983]). However, the assumption upon which this proposal was based, namely that the absence of esterase D activity in this case implied loss of both esterase D and RB genes, has been disproved (Hum. Genet., 76:33-40 [1987]). Nonetheless, the other findings show that chromosome 13 markers which were heterozygous in somatic cells often became homozygous or hemizygous in retinoblastoma tumors, and that there are homozygous deletions in the 13q14 region in 3/37 retinoblastoma cell lines (Nature, 305:779-784 [1983]; Proc. Natl. Acad. Sci., 83:7391-7394 [1986]). These experiments provide evidence that the proposed RB gene indeed functions in a recessive manner at the cellular level (Science, 235:305-311 [1987]; Cancer Res., 46:1573-1580 [1986]) in distinction to the "dominant" activity of classical oncogenes (Science, 228:669-676 [1985]; Nature, 315:190-195 [1985]) as measured, for example, by transfection assays.
Thus, while an extensive research is devoted to inherited and acquired retinoblastoma and to its causes, the reliable method for diagnosing the embryo, fetus, or newborn baby for the predisposition to inherited retinoblastoma, or the child or adult for their susceptibility to development of acquired retinoblastoma, or the secondary tumors often accompanying retinoblastoma, is still not readily available. That is true mainly because the protein intermediating the function of the RB gene has not been known, isolated or identified.
Therefore, it would be advantageous to acquire more knowledge about the molecular and chemical properties of the retinoblastoma gene such as the gene sequence, identification of the gene's location, its cloning and the isolation. Isolation of the RB gene's protein product and the identification of its amino acid sequence would also be advantageous as well as preparation of specific anti-RB protein antibody, particularly because the RB gene function is intermediated by the specific RB protein which, in turn, can only be recognized in the tissue by the specific anti-RB protein antibody.
Both forms of retinoblastoma can now be treated and most patients can be followed through adult life. However, patients with hereditary retinoblastoma have an extraordinarily high risk for developing a second nonocular malignancies (Ophthalmology, 91:1351-1355 [1984]; New Engl. J. Med., 285:307-311 [1971]; Cancer, 34:2077-2079 [1974]; up to 90% incidence within 30 years of initial diagnosis (Ophthalmology, 91:131-136 [1984]). The most frequently occuring secondary cancer is osteosarcoma, which is otherwise uncommon. In contrast, cured nonhereditary retinoblastoma patients show the same cancer rates as the general population. This finding is of considerable interest, since it implies that the RB gene may have a critical role in regulating other tumors as well.
Without complete isolation and identification of the RB gene and its protein product, an early diagnosis of the osteosarcoma or other RB secondary tumors, or the predisposition thereto, is unavailable.
Therefore, it would be advantageous to have available a means to diagnose the primary retinoblastoma or the susceptibility thereto, and to provide timely forewarning against the secondary molecular malignancies. The best way to achieve the above goals, is the by the identification of the amino acid sequence of the RB gene product protein, which protein, per se, is the intermediator of the RB gene regulatory function not only for retinoblastoma but also for its secondary tumorigenic regulating activity.
It is of obvious importance to understand the molecular nature of the RB gene, and the mechanism of its regulatory function through the protein product produced by retinoblastoma gene.
Recently, human retinoblastoma gene was successsfully cloned, identified and sequenced (Science 235:1394-1399 [1987]). The retinoblastoma gene was located in the chromosome 13 region 13q14:11 in the close proximity of the esterase D gene, also recently identified, cloned and sequenced (Proc. Natl. Acad. Sci., 83:6790-6794 [1986]; Proc. Natl. Acad. Sci., 83:6337-6341 [1986]). By chromosomal walking from esterase D gene, the retinoblastoma (RB) gene was identified on the basis of chromosomal location, homozygous deletion and tumorspecific alterations in expression. RB gene was shown to have 4723 nucleotides and encodes a messenger RNA (mRNA) of 4.8 kilobases (kb).
Transcription of RB DNA to RB mRNA was found to be abnormal in retinoblastoma patients. Transcription was either not detected at all, suggesting the absence or complete inactivation of the RB gene, or transcribed mRNA had shown decreased molecular size of about 4.0 kb, suggesting defective RB gene.
Sequence of RB complementary DNA (cDNA) clones yielded a single long open reading frame suggesting that it could encode a hypothetical protein of 816 amino acid. A computer-assisted search of a protein sequence data base revealed no closely related proteins suggesting a unique amino acid sequence of the predicted protein (Science, 235:1394-1399 [1987]). The predicted protein will seem to have several proline rich regions, similar to those previously observed in other nuclear oncogenes proteins such as proteins "myc" and "myb" (RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. [1985]).
The hypothetical amino acid composition of the RB gene product protein seems to contain several distinguishable regions which were shown to be similar to the other oncogenic proteins. This finding suggests that the RB gene protein product could have similar regulatory functions as these other oncogene proteins.
However, without chemical and molecular characterization on the RB gene protein product and without specific anti-RB protein antibody, the further elucidation of the RB protein in regulation of tumorigenesis is impossible.
Therefore, it would be advantageous to identify the amino acid sequence of the RB gene, to determine its subcellular localization and to determine whether it has DNA binding activity.
It would also be advantageous to obtain the exact amino acid sequence of the RB gene product, to prepare or isolate the portion or the whole RB protein in purified form, and to obtain a specific anti-retinoblastoma protein antibody which would specifically recognize the retinoblastoma protein in the tissue. Such antibody would then be employed as a diagnositc tool to recognize the presence or the absence of the RB gene product protein. Thus, the anti-RB protein antibody would diagnose the normal or abnormal protein of the RB gene in several different kinds of human cancer.
It is, therefore, the object of this invention to provide a complete amino acid sequence of the RB gene protein product.
It is another object of this invention to biochemically characterize the RB gene protein product and to determine its molecular weight.
It is still another object of this invention to determine the subcellular localization of the RB gene protein product.
It is yet another object of this invention to provide a metabolically labeled radioactive RB gene protein product.
It is a further object of this invention to provide a specific anti-retinoblastoma protein polyclonal antibody.
It is still a further object of this invention to provide a diagnostic method for hereditary predisposition to retinoblastoma in fetus, embryo and newborn, or for the susceptibility in the later age to acquire a secondary cancer associated with the retinoblastoma. Retinoblastoma gene involved cancers such as osteosarcoma, fibrosarcoma, glioblastoma and breast cancer.
It is still a further object of this invention to provide a method for treatment of cancerogenous growth through the regulation of growth promoting genes and by genetic manipulation.